The inward rectifying potassium (Kir) channel family consists of several potassium channels that play a central role in many cell types. These channels are notable for a unique cytoplasmic domain containing putative binding sites for regulatory ligands such as protons and membrane phospholipids. Mutations that alter responsiveness to these ligands can result in disease, e.g. Bartter's syndrome secondary to mutations in Kir1.1 that alter sensitivity to either pH or to phosphatidylinositol bisphosphate (PIP2), resulting in decreased channel activity and derangement of salt transport/excretion in the kidney. Recently, the crystal structure of one member of the prokaryotic protein family, KirBad.1, with significant primary sequence similarities with eukaryotic Kirs, was solved. We have shown that purified KirBad.1 reconstituted in liposomes is a potassium selective channel, regulated by protons and PIP2 with functional characteristics similar to eukaryotic Kirs. The unique ability to examine a pure protein preparation in a fully defined milieu, in combination with the crystal structure, provides a valuable tool in elucidating the structural mechanisms of ligand gating. The overall goal of this proposal is to define structural mechanisms of KirBac1.1 proton and phospholipid gating, using electrophysiological and liposomal assays. Results from this proposal will provide insight into proton and PIP2 gating mechanisms of Kir channels in general, and may lead to therapy of diseases caused by altered ligand regulation of Kir function, such as Bartter's syndrome.